75，No. 1（2007） 13

1 IntroductionTNT is an important explosive compound and itsdetection is highly important in many areas includingsecurity and health of people, environmental and toxico-logical effects, landmine search, aviation security andprevention of terrorist attacks.1-3）TNT is a prime con-stituent of the majority of landmines and bombs used bymilitary and terrorist forces around the world. It isbelieved that approximately 100 to 120 million of land-mines are scattered around the world which provideimmeasurable troubles such as ending of life, inflictinginjuries, spread of diseases, wastage of agricultural lands,poor economy, isolation and starvation, which obstacleeconomic revitalization in developing countries.1-6）Theenvironmental protection agency (EPA) considers TNTas toxic because it presents carcinogenic and mutageniceffects to all forms of life.6）The drinking water criterionfor TNT at a lifetime exposure cancer risk level is 1.0ppb. The most notable problems associated with theingestion of TNT are liver damage, gastritis, anemia,cyanosis, dermatitis and anorexia. Once a landmine isdeployed, TNT undergoes complex microbial and photo-chemical degradation, producing a number of degradedproducts. These signature compounds are typicallyreleased into the surrounding environment by severalmeans (diffusion through mine casing, leakage throughcracks or seams etc.) resulting in contamination of soiland ground water with TNT and its derivatives.7-9）The

extent of contamination depends on number of factorssuch as mine type, soil type, the moisture content, rainfall, wind flow and temperature variation of the soil.9）

Consequently, the concentration of TNT varies signifi-cantly from site to site and is expected that the concen-tration of TNT in soil or ground water is extremely low(parts-per-trillion (ppt) to parts-per-quadrillion (ppq)range). Thus, global security, environmental and lawenforcement agencies demand simple, highly sensitiveand selective detection methodologies for rapid and real-time detection of TNT. Nearly all of the instrumental methods (ranging frommetal detectors to bacteriological methods) have beeninvestigated for environmental analysis of TNT and/ortheir degradation products.1, 2, 4, 10-20）As a matter of fact,dog’s nose is the most sensitive and versatile sensorwhich can detect very faint signatures of TNT and itsrelated chemicals.12, 21）However, the use of dogs is limit-ed by several factors such as extensive training, behav-ioral variation and fatigue. Consequently, there is a greatinterest in the development of new-generation sensingtools which mimic dog’s nose without having their draw-backs. The most promising option in this respect wouldbe the SPR based immunosensors which can simultane-ously solves the problems of sensitivity, selectivity andspeed required for real-time detection of TNT.20, 22-24）

Immunosensors utilize high affinity antigen-antibodyrecognition for detection of target analytes.25, 26）SPR is a

― Review―

High-performance Surface Plasmon Resonance Immunosensors forTNT Detection

Norio MIURA,a＊ Dhesingh Ravi SHANKARAN,a, b Toshikazu KAWAGUCHI,aKiyoshi MATSUMOTO,c and Kiyoshi TOKOd

aArt, Science and Technology Center for Cooperative Research, Kyushu University (Kasuga-shi, Fukuoka 816-8580,Japan)bJapan Society for the Promotion of Science (Tokyo102-8471, Japan)cGraduate School of Agriculture, Kyushu University (Fukuoka 812-8581, Japan)dGraduate School of Information Science and Electrical Engineering, Kyushu University (Fukuoka 819-0395, Japan)

Received August 26, 2006 ; Accepted October 25, 2006

Detection of 2,4,6-trinitrotoluene (TNT) is an important environmental, security and health concern for the globalcommunity. TNT is a prime constituent of most of the landmines and bombs and is highly toxic and mutagenic.Various military and terrorist activities (e.g., manufacturing, waste discharge, testing and training) have resulted inextensive contamination of soil and ground water by TNT and its derivatives. Consequently, the development andapplication of new sensing techniques for detection and quantification of TNT has grown steadily over the years.Despite wide variety of analytical techniques, surface plasmon resonance (SPR) based immunosensors receivedgreat attention as a promising mean for TNT detection due to their advantages including high sensitivity, selectivi-ty, good versatility and high throughput. This review explores the recent trend and advancements in immuno-chemical techniques for environmental monitoring and field detection of TNT. The advantages of the surface plas-mon resonance as an optical signal transduction and indirect competitive immunoassay as the sensing principle arediscussed with special emphasis on our investigations on TNT detection. A brief description on explosives, land-mines and the current detection techniques (bulk and trace detection) is also provided.

Key Words : Immunosensors, Explosives, Landmine, TNT, SPR, Indirect Competitive Immunoassay

14 Electrochemistry

powerful optical signal-transduction tool for monitoringof binding event between biomolecules in real-time with-out the need of intrinsic or extrinsic labels.27, 28）Theadvantages of the SPR immunosensors with respect tosensitivity, selectivity, response time, multi-analyte detec-tion, on-field analysis, versatility and flexibility renderthem a promising alternative to traditional chromato-graphic and spectroscopic methods for efficient detectionof explosive compounds. We have been developing avariety of high performance SPR immunosensor. In thisreview article, we describe the recent trend and scope ofthe SPR immunosensors for environmental monitoring aswell as field detection of TNT.

2 Explosives and LandminesAn explosive is a material that under the influence ofthermal or mechanical shock, decomposes rapidly andspontaneously with the evolution of a large amount of

heat and gases. In general, explosives are classified intoprimary and secondary ones based on their susceptibilityto initiation. Primary (or initiating) explosives are verysensitive and susceptible to ignition (e.g., lead azide, leadstyphnate, mercury fulminate). They are often used insmall quantities to ignite (set off) secondary explosives.Secondary (or military) explosives are formulated to det-onate under specific circumstances. They are less sensi-tive to mechanical or thermal shock, but explode withgreat violence when set off by an initiating explosive.Secondary explosives constitute major part of the land-mines, which mostly include nitroaromatics, nitraminesand nitrate esters. Table 1 presents the physical andchemical properties of the military explosives commonlyused in landmines.29, 30）Among them, TNT is the mostwidely used military explosive in antipersonnel and anti-tank landmines and has been in use for about the last100 years (mostly used during World War I and II).

Table 1 Physiochemical properties of important secondary (military) explosives.

75，No. 1（2007） 15

Depending on the purpose, various combinations of thesemilitary explosives are used along with a number oforganic compounds (waxes, plasticizers, stabilizers, oilsetc.) while preparing landmines.

3 Analytical Methods for TNT DetectionThere are a variety of technologies employed fordetection of TNT and its signature compounds as givenin Fig. 1. The analytical methods for detection of explo-sives can be broadly classified into trace and bulk detec-tion. Trace detection includes chemical identification ofexplosive residue in either vapor or liquid phase. Bulkdetection methods are usually applied to security (air-port luggage or mail screening) or nondestructive test-ing. Metal detectors, mechanical methods and dogs aremost commonly used in field detection of landmines.Recent trend in the use of plastic-cased mines with fewor no metal parts has made the detection process veryslow and difficult with even advance electromagneticinduction (EMI) and ground penetrating radar (GPR).Moreover, they have high false positive rates due to thedifficulty in discrimination between mines and otherburied objects. Mechanical methods are very labor inten-sive and can be quite dangerous. The use of dogs fordetection of explosives is well established (police forces,military and humanitarian agencies), but dogs are limitedby extensive training costs, behavioral variation andfatigue. In addition, a number of other detection tech-niques (nuclear, chromatographic and spectroscopic) arebeing in practice. The strength and drawbacks of various analyticalmethods for detection of TNT are exemplified by severalarticles.1, 2, 4, 10-20, 30-36）Reviewing the literature, it is gener-ally accepted that despite good sensing capabilities, mostof these technologies have practical limitations such asslow response, variation of results with weather condi-tions and background environment, less effective todeeply buried landmines, complicated and time consum-ing procedures, bulky and expensive instrumentation.Thus, active research is being pursued for the develop-ment of simple and innovative sensors, which are sensi-tive, specific, rapid and cost-effective for on-site detectionof explosives. Immunosensor technology is one of thepromising approaches in this respect, because it is recog-nized as a high throughput analytical tool for identifica-tion and quantification of analytes in complex biologicaland environmental samples with high specificity and sen-sitivity within a short detection time.22-28）

4 Immunosensor for TNT DetectionImmunosensors utilize high-affinity recognition ofimmobilized antibodies for the detection of antigens orvice-versa, which in combination with a suitable trans-ducer technology enables sensitive and selective analysisof TNT in complex environmental samples. A number oftransducers such as piezoelectric, optical and electro-chemical devices are used for the construction ofimmunosensors for TNT detection, among which opticaltransduction was widely used. Furthermore, TNT quan-tification has been carried out by using different assay

formats such as direct, indirect competitive, sandwichand displacement.22-24, 37-56）A brief introduction to some ofthe immunoassays demonstrated for TNT detection isgiven in Table 2.In general, transduction of affinity biosensors can bedone in two ways, i.e., labeled and label-free approaches.If labeled species are employed, the analyte quantifica-tion can be inferred from the number of labeled mole-cules which bind to (or desorbed from) the transductionsurface. Until recently, most of the immunoassays report-ed for TNT detection have relied on labeling of the anti-bodies or antigens (such as enzyme immunoassay,radioimmunoassay and fluorescent immunoassay).40-54）Inspite of the fact that some of these methods are sensi-tive, most of them have difficulties owing to the multi-step procedures for sensor fabrication and labeling of thereagents. It is believed that labeling of the reagents mayalter the activity of the labeled-biomolecules, resulting indegrading performances. Hence, it is important to havelabel-free immunochemical methods for efficient quantifi-cation of TNT. One of the promising options for label-free detection ofexplosives is the use of SPR as the signal transduction.SPR is an optical spectroscopic technique, which pro-vides sensitive, fast and real-time information on biomole-cular interactions even without the need for labeling ofthe reagents.

5 SPR immunosensors - principle and operationSPR is a surface sensitive spectroscopic technique,which has been successfully implemented into animmunosensor platform for simple, rapid and label-freeanalysis of various biomolecular interactions. The princi-ple and application of the SPR technique has beendemonstrated extensively in several review papers.25-28, 57）

Fig. 1 Analytical methods employed for detection of TNT.

16 Electrochemistry

A simple schematic view of the operation principle ofSPR immunosensors is given in Fig. 2. SPR takes advan-tage of the resonance coupling between the excitingradiation and the surface plasmons waves propagatesalong the surface of a thin metal (gold) film for probingchanges near the metal surface. The sensitive nature ofthe SPR sensor is derived from the unique characteris-tics of the SPR-generated evanescent field at themetal/dielectric interface under the total internal reflec-tion condition. The linear relationship between the SPRsignal and the refractive index and/or thickness changesenables SPR to measure accurately the adsorption of themolecules on the metal surface and their eventual inter-actions with specific ligands. This change can beobserved via the shift in the resonance angle of the SPRcurve. This simple technique finds application in variousresearch fields including drug discovery, food analysis,environmental monitoring, defense and biowarfare detec-

tion, molecular engineering and cell biology. A majoradvantage of SPR is that it offers a label-free and nonin-vasive method to perform real-time analysis of surfacereactions, which can be also utilized for efficient quantifi-cation of TNT.

6 SPR Immunoassay Formats for TNTDetection

Although SPR is capable of detecting the biomolecularinteraction with high sensitivity, it has difficulties withthe direct detection of low concentrations of small mole-cules (molecular weight (MW)＜ ca . 1000 Da). Theincrease in the refractive index produced by the adsorp-tion of low-molecular-weight analyte is often not suffi-cient enough to be detected directly. Since TNT is asmall molecule (MW＝227.13), it is hard to detect lowconcentrations of TNT by direct SPR method. However,the sensitivity of the SPR technique can be enhanced byutilizing different assay principles. Both sandwich anddisplacement assays generally require labeling of theregents. In displacement assay format, excess of labeledanalyte is introduced over an antibody immobilized sur-face to occupy all of the binding sites of the antibody.Upon introduction of unlabeled analyte, displacement ofthe labeled analyte occurs and is measured by fluores-cence or chemiluminescence method.50-54）Despite wideuse of label-based displacement assay format for TNTdetection, it is rarely employed in SPR based label-freeassays. Very recently, we have shown the possibility ofusing SPR based displacement assay for label-free detec-tion of TNT.58）Sandwich assays require relatively largeantigens that contain at least two epitopes for antibodybinding. The antigen is bound to the immobilized cap-ture antibody at one epitope and is detected by using afluorescently-labeled tracer antibody bound to a secondepitope. Since TNT is a small molecule, it is hard todetect by sandwich assay format. Hence, reports on SPRbased label-free sandwich assays are limited for TNTdetection.55）

In order to solve the limits of anlayte size imposed onSPR-based direct detection methods and labeling withsandwich and displacement methods, we employed thecompetition-based indirect immunoassay for the detec-

Fig. 2 Schematic view of the operation principle of SPRimmunosensor.

Table 2 Immunoassays developed for detection of TNT.

75，No. 1（2007） 17

tion of TNT. It has been widely accepted that the princi-ple of indirect competitive inhibition can greatly bolsterSPR’s ability to detect very low concentrations of low-molecular-weight analytes.59-61）It is worthy to note herethat the principle of indirect competitive inhibition wasfirst introduced by our group in 1992 to enhance the sen-sitivity of a label-free detection of low-molecular-weightanalytes (methamphetamine, MW＝149) in quartz crys-tal microbalance (QCM) based immunoassays.62, 63) Theadvantage of this assay format has been successfullyextended to SPR signal transduction for detection of avariety of small analytes of biomedical and environmen-tal interests by our group as well as by several otherresearch groups around the world.22 -24 , 59 -61 , 64 -69）

Considering the need for highly sensitive analytical sens-ing system for TNT monitoring and the advantage ofthe SPR immunosensors, we have developed several SPRimmunoassays for detection of TNT and its derivativesbased on the principle of indirect competitive immunore-action.20, 22-24, 37, 61, 70-73）

7 Principle of Indirect Competitive InhibitionSolid-phase immunoassays generally consists of anantibody or antigen immobilized on a transducer surfaceand results in a signal generated from the binding inter-action between the antigen and the antibody at the sen-sor surface. It is known that the antigen immobilizedsurface has higher stability and reproducibility than theantibody immobilized surface and has been mostlyemployed in competitive-based detection. In this method,an analyte is incubated together with antibody and themixture is allowed to flow over a conjugate-immobilizedsurface for binding of free antibody. Figure 3 representsthe schematic view of the principle of indirect competi-tive immunoreaction.37）By using TNP-BSA conjugate(antigen) and anti-TNT antibody, the principle of indirectcompetitive inhibition can be explained as follows. TNP-BSA conjugate is immobilized onto an SPR-gold surfaceand anti-TNT antibody is introduced in a continuousflow over the conjugate surface. A large increase in theresonance angle (∆θ0) occurs when antibody binds withTNP-BSA conjugate immobilized on an SPR gold chip.However, when an equilibrium mixture of antibody andTNT (analyte) is let to flow over the conjugate, only theunbound antibody in the equilibrated mixture can beavailable for binding with the conjugate surface, andthe resonance angle shift will be decreased to (∆θ). Thechange in the resonance angle shift will decrease withincreasing concentration of TNT (analyte). The mea-sured binding response (∆θ) is therefore correlatedinversely to the concentration of free TNT in solution.Based on this correlation, concentration of TNT can bedetermined. After each immunoreaction, the conjugatesurface can be regenerated by dissociating the antigen(conjugate)-antibody immunocompound by means offlowing of suitable eluents (e.g. pepsin solution).

8 Experimentation for TNT DetectionThe immunoassay experiments were mostly carriedout with an SPR 670 instrument (Nippon Laser and

Electronics, Japan) at a room temperature of 25±1℃.Biacore J-2000 SPR instrument (Uppsala, Sweden) wasalso used in some studies. ELISA measurements wereperformed using 96-well immunoplates (NUNC, No.446612, Denmark) and a microplate reader (Spectra 1,Wako, Japan). The gold chips were prepared in our labo-ratory by vacuum sputtering of high purity gold overBK7-type microscopic glass slides. Phosphate bufferedsaline (PBS, 0.1 M, pH 7.2, 1 vol％ ethanol) was used as acarrier solution. A variety of commercial and home-made conjugatesand antibodies were used in our studies for detection ofTNT and its derivatives. The typically used conjugatesare; 2,4,6-trinitrophenol-bovine serum albumin (TNP-BSA), TNP-ovalbumin (TNP-OVA), 2,4,6-trinitrophenyl-OVA (TNPh-OVA), TNPh-keyhole limpet hemocyanine(TNPh-KLH). The monoclonal (M) and polyclonal (P)antibodies (Ab) used are: M-TNT Ab (StrategicBiosolutions, USA), M-TNP Ab (Biomeda, USA), P-TNPAb (home-made and BD Biosciences, USA), P-TNPh-KLHAb (home-made). TNT solution was obtained fromChugoku Kayaku, Co., Ltd. Japan.

. 9 Detection of TNT by Indirect competitive

SPR Immunoassay There are several key factors involve in the develop-ment of SPR-based indirect immunoassay for detection ofTNT such as preparation of biomolecules, sensor fabrica-tion, immunoreaction, nonspecific adsorption, surfaceregeneration, cross-sensitivity and stability. The qualityand quantity of these factors plays a central role in thefinal performances of the immunoassays for TNT detec-tion. The following sections provide an introduction tothese features according to our experiences in the con-struction of SPR immunosensors for TNT detection. 9.1 Preparation of conjugates and antibodies The sensitivity and selectivity of an immunosystem isstrongly dependent on the nature and recognition capa-bility of antibody towards its target analyte (antigen).There are several commercial conjugates and antibodiesavailable for TNT and its derivatives. Since TNT is asmall molecule, it should be coupled with a carrier pro-

Fig. 3 Schematic view of the principle of indirectcompetitive immunoreaction. Adopted with permission fromref. 37.

18 Electrochemistry

tein in order to introduce an antibody response in a ver-tebrate immune system as well as for effective immobi-lization onto a gold surface. The usual protein carriersare BSA, OVA and KLH. In addition to the commerciallyavailable conjugates and antibodies, we have preparedseveral conjugates with these carrier proteins.22-24, 70, 71）

Few antibodies were also prepared by use of these con-jugates.20, 22, 24, 37, 70, 71）The conjugates are generally pre-pared by reacting 2,4,6-trinitrobenzene sulfonate sodiumsalt with NaHCO3 solution (pH 8.5) containing respectivecarrier protein for 2 h at 40℃. After the reaction, theresulting conjugates were dialyzed in H2O at 4℃ for 2days and lyophilized. The conjugates thus preparedwere used as immunogens for obtaining antibodies. Forexample, Fig. 4 depicts the procedure used for prepara-tion of P-TNPh-KLH antibody by immunization of rabbitswith TNPh-KLH conjugate. We observed that the conju-gate and the antibodies prepared with KLH (a proteinfrom mollusks) gave better performances possiblybecause of its higher molecular mass and strongerimmunogenicity. 9.2 Fabrication of immunosensorImmobilization of a recognition element onto a trans-ducer surface is a prime factor in immunosensor designand has long been realized that simple, rapid and reliableimmobilization route is the key to successful fabricationof commercially viable sensors. There are several immo-bilization chemistries being practiced for sensor fabrica-tion, such as physical adsorption, self-assembling, embed-ding in polymers or membranes, Langmuir-Blodgettdeposition, sol-gel process etc., each having their ownadvantages and limitations.25-28）Among them, physicaladsorption is the simplest method and has the advantageof immobilizing large amount of biomolecules on thetransducer surface (gold). Considering the fact thatimmobilization of the analyte derivative is more pre-ferred due to its stability compared to the immobilizedantibody, we prepared immunosensor surfaces by immo-bilization of protein-based conjugates on gold surfaces bythe physical adsorption method.20, 22-24, 71）Figure 5 illus-trates the fabrication of an immunosensor by physicaladsorption of TNP-OVA conjugate onto an SPR-gold sur-face. We observed that the monomolecular layer of thephysically immobilized conjugates is highly active,robust and regenerable. It is believed that the conjugatesare uniformly immobilized on the gold surface in a two-dimensional arrangement. In addition, we also used theself-assembling technique for fabrication of immunosen-sor surface using poly (ethylene glycol) (PEG) thiolate asa functional monolayer linker-molecule. The PEG basedself-assembled immunosensor surface was found to bestable and highly resistant to non-specific protein adsorp-tion. Non-specific protein adsorption is an important factorin SPR immunoassays and is critical to have a proteinnon-fouling background during the analyses of real envi-ronmental samples. Otherwise, it may cause error in theSPR measurement due to the adsorption of irrelevantchemicals and proteins on the hydrophobic transducersurfaces. In our studies we allowed excess of BSA pro-

tein (ca. 1000 µg/ml) to flow over the physically-immobi-lized conjugate surface, which occupies any of theadsorption sites left available (unoccupied by the conju-gate) on the SPR-gold surface (Fig. 5).23）

9.3 Regeneration of the sensor surfaceIn immunoassays, it is preferable to have highly regen-

erable surface because surface regeneration is a key fac-tor to reduce cost and to perform multiple measure-

Fig. 4 Schematic view of the procedure for anti-TNPh-KLH antibody preparation.

Fig. 5 (A) SPR sensorgram for immobilization of TNP-OVA conjugate (curves a and b) and BSA (curves c and d)onto an SPR gold surface. Carrier solution: PBS, flow speed:10 µl/min for TNP-OVA injection, 15 µl/min for BSAinjection. (B) Plot of the SPR angle shift against theconcentration of TNP-OVA conjugate. Adopted withpermission from ref. 23.

75，No. 1（2007） 19

ments speedy and accurately. After an immunoreaction,the binding sites of the conjugate can be regenerated bydissociating antigen-antibody immunocompound usingdifferent eluents (HCl, NaOH, glycine-HCl, pepsin,ethanolamine etc.) in the pH range from highly acidic tobasic.64-71）In our experiments, we mostly used pepsinsolution (0.2 M, prepared with glycine-HCl buffer at pH2.0) for surface regeneration.23, 37, 71）In addition, glycine-HCl and NaOH were also used.24）The selection of aneleuent depends on the nature and strength of animmunoreaction. Figure 6 shows the SPR response forimmunoreaction and surface regeneration of four differ-ent antibodies with a TNPh-OVA conjugate surface. It isseen from the figure that in the case of polyclonal anti-bodies, higher concentration of pepsin is used for surfaceregeneration compared with monoclonal antibodies. Theregeneration conditions indicate that the binding of theP-TNPh Ab with the conjugate is much stronger thanother antibodies. It is possibly because of high affinity ofthe P-TNPh Ab as well as the multivalent protein bind-ing to the conjugate due to its polyclonal behavior. It isexpected that the association of monoclonal antibodieswith conjugates involves selective monovalent attach-ment, which can be easily dissociated by the use of lessconcentration of pepsin compared to polyclonal antibod-ies.Stability of an immunosensor is an important issue fordeciding its applicability to practical repeatable analysis.In general, stability of an immunosensor is decided byseveral factors such as nature of the biomolecule, immo-bilization methods, regeneration conditions etc. Most ofour immunosensors showed good stability and repro-ducibility despite fabrication by simple physical adsorp-tion. The immunosenors retained their original activitiesfor more than 30 immunocycles. Recently, we studied thedetection of TNT with a PEG based self-assembledimmunosensor, which showed excellent stability for notless than 100 cycles (Fig. 7). 9.4 Indirect competitive detection of TNT For detection of TNT by indirect competitiveimmunoassay, standard TNT solutions were preparedwith PBS solution containing fixed amount of antibodiesand subjected to incubation for 10 min. The TNT pre-senting in solution competes with the immobilized conju-gates for binding sites on the antibodies, which result ina concentration-dependent change in the resonanceangle shift in the detectable range of TNT. Figure 8shows the SPR response observed for indirect competi-tive detection of TNT based on the immunoreactionbetween M-TNT Ab and TNPh-KLH conjugate immobi-lized on a gold surface.71）Antibody solutions containingdifferent concentrations of TNT were allowed to flowover the TNPh-KLH conjugate at a rate of 100 µl/min(flow duration＝36 s). It was observed that the reso-nance angle shift decreased progressively with increas-ing concentration of TNT, as shown in the figure. This isconsistent with the fact that TNT in solution associateswith M-TNT Ab and inhibits the antibody from bindingwith TNPh-KLH conjugate. There is no appreciablechange in the resonance angle for the flow of BSA pro-

tein in the middle of the immunoassay, which clearlyindicates that the conjugate surface was not degradedduring the immunoassay (even by the surface regenera-tion process using pepsin solution). The above resultssuggest that the immunoassay is remarkably sensitiveand the sensor surface is highly robust and regenerable.Moreover, the response time is only 36 s and a singleimmunocycle could be done within 2 min including sur-face regeneration. This enables rapid detection of TNT. From the results of the indirect immunoassay studies,dose-response curves were plotted between the percent-age of inhibition and the logarithm of TNT concentra-tion. The dose-response curve is usually sigmoidal inshape for indirect competitive SPR immunoassays. Thelimit of detection is the concentration of TNT whichgives a minimum detectable difference signal that isequal to 3 standard deviation of the mean response ofthe blank sample (zero TNT concentration). In most ofour immunosystems, the working range is definedbetween 15％ (upper detection limit) and 85％ (lowerdetection limit). Within this range, a near linear correla-tion is seen between the changes in the resonance angleand the TNT concentration. Figure 9 illustrates the dose-response curve observed the four different antibodieswith TNPh-KLH conjugate. The error bars represent themean of triplicate samples at each dilution. As can be

Fig. 6 SPR response transients observed for theimmunoreaction of TNPh-OVA conjugate with P-TNPh-KLH Ab (curves a and b), P-TNP Ab (c), M-TNT Ab (d) andM-TNP Ab (e). Carrier solution: PBS, flow rate: 100 µl/min.Adopted with permission from ref. 22.

Fig. 7 The stability of the immunoreaction between TNTimmobilized PEG-NH2 SAM surface and M-TNT Ab onmultiple cycles. Carrier solution: PBS, flow rate: 100 µl/min,flow duration: 2 min. Unpublished result.

20 Electrochemistry

seen from the figure, among the four antibodies, the P-TNPh Ab showed a highest sensitivity in the concentra-tion range from 0.002 ng/ml to 150 ng/ml with a detec-tion limit of 0.002 ng/ml (2 ppt) followed by M-TNT Ab,which exhibited a detection range from 0.008 ng/ml to400 ng/ml. The high sensitivity observed with P-TNPhAb is possibly due to its higher affinity for TNT com-pared to other antibodies. It is noteworthy that the sensi-tivity given by the commercial M-TNT Ab in the pre-sent label-free SPR assay is higher than the other label-based immunosensors reported with the same antibody(monoclonal anti-TNT Ab from Strategic Biosolutions) indifferent assay formats such as ELISA,40, 45）direct,41）indi-rect,41）and displacement immunoassays.19, 41, 51）Similarimmunoassay experiments were performed with differ-ent combination of conjugates and antibodies. The lineardynamic range observed with several immunosystemsdeveloped in our laboratory is illustrated in Fig. 10. 9.5 Cross-sensitivity and matrix effectExplosive samples are complex in nature and hencethe matrix effect and cross-sensitivity from the non-tar-get materials is a major concern in real-time monitoringof TNT. Cross-reactivity is the degree to which an anti-body will bind to a substance other than its target and isprimarily depends on the nature and affinity of the anti-body towards the target and the non-target analytes. Wehave evaluated the cross-sensitivity for most of theimmunosystems against a variety of structural analoguesof TNT.23, 24, 70, 71）The cross-sensitivity is calculated basedon the analyte concentration yielding 50％ inhibitionaccording to the formula (C0/C)×100, where C0 is theconcentration of TNT at 50％ inhibition and C is theconcentration of the cross-reacting analyte at 50％ inhi-bition. Table 3 shows the cross-sensitivity profile for twoimmynosystems in the detection of TNT. It is generallybelieved that polyclonal antibodies show more cross-reac-

tivity compared to monoclonal antibodies, but is interest-ing to note that similar to M-TNT Ab, the P-TNPh Abdemonstrated low cross-reactivity to many of the struc-turally related compounds, while demonstrating highaffinity for the target TNT. The cross-reactivity is oftennot a disadvantage, because it permits the use of theassay for the rapid screening of a family of explosivesenabling detection of landmines.

10 Gas-phase Detection of TNT by IndirectCompetitive SPR Immunoassay

Gas-phase detection of TNT is highly important espe-cially to identify the presence of explosives in airportscreening. In addition to the liquid phase detection ofTNT, attempts are being made to detect gas-phasedetection of TNT by our group. Vapors of TNT and itsderivatives were preconcentrated into PBS buffer andwere analyzed based on indirect competitive SPRimmunoassay method.73）Figure 11 depicts a simpleschematic process of preconcentration of TNT for detec-tion by SPR. In the initial attempt, we have achieved adetection limit down to ppb level. The proposed systemhas promising scope for further lowering of the detectionlimit.

Fig. 8 SPR response of a TNPh-KLH conjugateimmobilized gold-chip to the flow of 10 µg/ml M-TNT Ab inthe absence (a) and in the presence of 0.01 ng/ml (b), 1ng/ml (c) and 100 ng/ml (d) TNT solutions. Curve (e)corresponds to the flow of 200 µg/ml PBS. Carrier solution:PBS, flow speed: 100 µl/min, flow duration: 36 s. Adoptedwith permission from ref. 71.

Fig. 9 Dependence of the percentage of inhibition on theconcentration of TNT with four immunosystems. Adoptedwith permission from ref. 71.

Fig. 10 Linear dynamic range for the detection of TNTwith various immunosystems developed in our laboratory.

75，No. 1（2007） 21

11 ConclusionsIn the era of international terrorism and weapons ofmass destruction, it is important to have high perfor-mance explosive sensors to promote safety, security andhealth of human and eco system. We have demonstratedthe potential of SPR-based indirect competitive assay fordetection of TNT with significant analytical characteris-tics. Our SPR immunosensors are ultra-highly sensitivedown to 1 ppt level with dynamic ranges covering fourorders of magnitude. These label-free immunoassays arehighly regenerable and rapid, taking only couple of min-utes to complete a single measurement including surfaceregeneration. Due to these advantages, our SPRimmunosensors provide a valuable alternative to conven-tional chromatographic and spectroscopic procedures forenvironmental monitoring and field detection of TNT.We believe that SPR immunoassays will undoubtly playan important role in future environmental monitoring ofexplosives and forefront of homeland security. In thiscontext, portable immunosytems which can representthe technological version of a dog’s nose for TNT detec-tion are need to be developed. Current research in ourgroup is progressing in this area in the development ofportable systems (hand-held and mobile devices) whichcould possibly be housed in soldier’s or farmer’s back-pack and in the hands of security persons in airports forrapid and high throughput monitoring of explosives.

AcknowledgementWe are thankful to Japan Science and TechnologyAgency for partial funding of this work (CREST -Development of ultra supersensitive biosensor for explo-sive molecules). One of the authors (D. Ravi Shankaran)gratefully acknowledges Japan Society for the Promotionof Science (JSPS), Tokyo, Japan for providing a post-doc-toral fellowship.

References1）J. Yinon, Trends Anal. Chem., 21, 292 (2002).2）A. M. Rouhi, Chem. Eng. News, 75, 1 (1997).3）S. S. Talmage, D. M. Opresko, C. S. Maxwell, C. J. E.Welsh, F. M. Cretella, P. H. Reno, and F. B. Daniel, Rev.

Environ. Contam. Toxicol., 161, 1 (1999). 4）J. A. MacDonald, Environ. Sci. Tech., 35, 370A (2001).5）T. A. Lewis, D. A. Newcombe, and R. L. Crawford, J.

Environ. Manag., 70, 291 (2004).6）Environmental Protection Agency; Health Advisory for

TNT. Criteria and Standard Division. Office of DrinkingWater, Washington DC (1989).

7）A. E. Nunez, A. Caballero, and J. L. Ramos, Microbiol.Mol. Biol. Rev., 65, 335 (2001).

8）T. F. Jenkins, D. C. Leggett, P. H. Miyares, M. E.Walsh, T. A. Ranney, J. H. Cragin, and V. George,Talanta, 54, 501 (2001).

9）S. W. Webb and J. M. Phelan, Proc. SPIE, 5089, 970(2003).

10）J. Yinon, Anal. Chem., 75, 98A (2003).11）G. A. Eiceman, Anal. Chem., 76, 390A (2004).12）K. G. Furton and L. J. Myers, Talanta, 54, 487 (2001).13）M. Nambayah and T. I. Quickenden, Talanta, 63, 461(2004).

14）A. M. Zoubir, IEEE Sens. J., 2, 41 (2002).15）E. M. A. Hussein and E. J. Waller, Appl. Rad. Isotopes,53, 557 (2000).

16）T. A. Lewis, D. A. Newcombe, and R. L. Crawford, J.Environ. Manag., 70, 291 (2004).

17）A. M. Jimenez and M. J. Navas, J. Hazard. Mater., 106A,1 (2004).

18）Y. Tan, L. G. Huettel, S. L. Tantum, and L. M. Collins,Surf. Sci. Tech. Appl., 4, 263 (2003).

19）P. T. Charles, J. G. Rangasammy, G. P. Anderson, T. C.Romanoski, and A. W. Kusterbeck, Anal. Chim. Acta,525, 199 (2004).

20）D. R. Shankaran, K. V. Gobi, T. Sakai, K. Matsumoto, T.

Fig. 11 Schematic view of the sampling process for gas-phase detection of TNT. Adopted with permission form ref.73.

Table 3 Cross-sensitivity of the nitroaromatic analogs forthe detection of TNT with two immunoassays. Adoptedwith permission from ref. 71.

P-TNPh Ab M-TNT Ab

AnalyteIC50[ng/ml]

CrossIC50[ng/ml]

Cross

sensitivity [%] sensitivity [%]

TNT 0.3 100 0.7 100

RDX 510 0.059 1420 0.049

HMX 970 0.031 1870 0.037

2,4-DNT 56 0.54 82 0.85

1,3-DNB 32 0.94 185 0.38

2A-4,6-DNT 75 0.40 42 1.67

4A-2,6-DNT 305 0.098 830 0.084

IC50＝Concentration of analyte at 50% of inhibition

22 Electrochemistry

Imato, K. Toko, and N. Miura, IEEE Sens. J., 5, 616(2005).

21）R. J. Harper, J. R. Almirall, and K. G. Furton, Talanta,67, 313 (2005).

22）D. R. Shankaran, K. Matsumoto, K. Toko, and N. Miura,Electrochemistry, 74, 141 (2006).

23）D. R. Shankaran, K. V. Gobi, T. Sakai, K. Matsumoto, K.Toko, and N. Miura, Biosens. Bioelectron., 20, 1750(2005).

24）K. Matsumoto, A. Torimaru, S. Ishitobi, T. Sakai, H.Ishikawa, K. Toko, N. Miura, and T. Imato, Talanta, 68,305 (2005).

25）P. B. Luppa, L. J. Sokoll, and D. W. Chan, Clin. Chim.Acta, 314, 1 (2001).

26）M. A. Cooper, Nat. Rev. Drug Discov., 1, 515 (2002).27）J. Homolo, Anal. Bioanal. Chem., 377, 528 (2003).28）C. L. Baird and D. G. Myszka, J. Mol. Recognit., 14, 261(2001).

29）M. E. Walsh, T. F. Jenkins, and P. G. Thorne, J. Energ.Mater., 13, 357 (1995).

30）C. Bruschini, Subsurf. Sens. Tech. Appl. 2, 299 (2001).31）D. S. Moore, Rev. Sci. Instr., 75, 2499 (2004).32）G. K. Kannan, R. Bhalla, J. C. Kapoor, A. T. Nimal, U.Mittal, and R. D. S. Yadava, Defence Sci. J., 54, 309(2004).

33）M. Altamirano, L. G. Villada, M. Agrelo, L. S. Martin, L.M. Otero, A. F. Moya, M. Rico, V. L. Rodas, and E.Costas, Biosens. Bioelectron., 19, 1319 (2004).

34）M. E. Walsh and T. Ranney, J. Chromatogr. Sci., 36, 406(1998).

35）T. Khayamian, M. Tabrizchi, and M. T. Jafari, Talanta,59, 327 (2003).

36）K. Masunaga, K. Hayama, T. Onodera, K. Hayashi, N.Miura, K. Matsumoto, and K. Toko, Sens. Actuators B,108, 427 (2005).

37）D. R. Shankaran, K. Matsumoto, K. Toko, and N. Miura,Sens. Actuators B, 114, 71 (2006).

38）J. L. Elkind, D. I. Stimpson, A. A. Strong, D. U.Bartholomew, and J. L. Melendez, Sens. Actuators B, 54,182 (1999).

39）A. Larsson, J. Angbrant, J. Ekeroth, P. Mansson, and B.Liedberg, Sens. Actuators B, 113, 730 (2006).

40）P. T. Charles, L. C. S. Lake, S. C. Francesconi, A. M.Churilla, J. G. Rangasammy, C. H. Patterson, J. R.Deschamps, and A. W. Kusterbeck, J. Immunol.Methods, 284, 15 (2004).

41）K. E. Sapsford, P. T. Charles, C. H. Patterson, and F. S.Ligler, Anal. Chem., 74, 1061 (2002).

42）R. Wilson, C. Clavering, and A. Hutchinson, Analyst,128, 480 (2003).

43）S. K. V. Bergen, I. B. Bakaltcheva, J. S. Lundgren, andL.C.S. Lake, Environ. Sci. Technol., 34, 704 (2000).

44）M. Alstein, A. Bronshtein, B. Glattstein, A. Zeichner, T.Tamiri, and J. Almog, Anal. Chem., 73, 2461 (2001).

45）A. Zeck, M. G. Weller, and R. Niessner, Fres. J. Anal.Chem., 364, 113 (1999).

46）P. Julicher, E. Mussenbrock, R. Renneberg, and K.Cammann, Anal. Chim. Acta, 315, 279 (1995).

47）I. M. Ciumasu, P. M. Kramer, C. M. Weber, G. Kolb, D.Tiemann, S. Windisch, I. Frese, and A. A. Kettrup,Biosens. Bioelectron., 21, 354 (2004).

48）I. B. Bakaltcheva, F. S. Ligler, C. H. Patterson, and L. C.S. Lake, Anal. Chim. Acta, 399, 13 (1999).

49）P. M. Kramer, E. Kremmer, C. M. Weber, I. M.Ciumasu, S. Forster, and A. A. Kettrup, Anal. Bioanal.Chem., 382, 1919 (2005).

50）T. M. Green, P. T. Charles, and G. P. Anderson, Anal.Biochem., 310, 36 (2002).

51）E. R. Goldman, G. P. Anderson, N. Lebedev, B. M.Lingerfelt, P. T. Winter, C. H. Patterson, and J. M.Mauro, Anal. Bioanal. Chem., 375, 471 (2003).

52）G. P. Anderson, S. C. Moreira, P. T. Charles, I. L.Medintz, E. R. Goldman, M. Zeinali, and C. R. Taitt,Anal. Chem., 78, 2279 (2006).

53）U. Narang, P. R. Gauger, A. W. Kusterbeck, and F. S.Ligler, Anal. Biochem., 255, 13 (1998).

54）P. R. Gauger, D. B. Holt, C. H. Patterson, P. T. Charles,L. S. Lake, and A. W. Kusterbeck, J. Hazard. Mater., 83,51 (2001).

55）P. Pfortner, M. G. Weller, and R. Niessner, Fres. J. Anal.Chem., 360, 192 (1998).

56）C. Heiss, M. G. Weller, and R. Niessner, Anal. Chim.Acta, 396, 309 (1999).

57）D. R. Shankaran, K. V. Gobi, and N. Miura, Sens.Actuators B, available on line.

58）T. Onodera, K. Harada, K. Horikawa, P. Singh, N.Miura, K. Matsumoto, and K. Toko, 11-IMCS-Proceedings, p.226 (2006)

59）N. Miura, K. Ogata, G. Sakai, T. Uda, and N. Yamazoe,Chem. Lett., 26, 713 (1997).

60）N. Miura, M. Sasaki, K. V. Gobi, C. Kataoka, and Y.Shoyama, Biosens. Bioelectron., 18, 953 (2003).

61）D. R. Shankaran, K. V. Gobi, K. Matsumoto, T. Imato,K. Toko, and N. Miura, Sens. Actuators B, 100, 450(2004).

62）N. Miura, H. Higobashi, G. Sakai, A. Takeyasu, T. Uda,and N. Yamazoe, Technical. Digest, 4th International meet.Chem. Sens., Tokyo, p.228 (1992).

63）N. Miura, H. Higobashi, G. Sakai, A. Takeyasu, T. Uda,and N. Yamazoe, Sens. Actuators B, 13, 188 (1993).

64）G. Sakai, S. Nakata, T. Uda, N. Miura, and N. Yamazoe,Electrochim. Acta, 44 3849 (1999).

65）K. V. Gobi, H. Tanaka, Y. Shoyama, and N. Miura, Sens.Actuators B, 111-112, 562 (2005).

66）S. Kumbhat, D. R. Shankaran, S. J. Kim, K. V. Gobi, V.Joshi, and N. Miura, Chem. Lett. 35, 678 (2006).

67）S. J. Kim, K. V. Gobi, R. Harada, D. R. Shankaran, andN. Miura, Sens. Actuators B, 115, 349 (2006).

68）T. Onodera, R. Harada, D. R. Shankaran, T. Sakai, J.Liang, K. Matsumoto, N. Miura, T. Imato, and K. Toko,Systems and Human Science-for Safety, Security andDependability, Elsevier, p.287 (2005).

69）Q. Yu, S. Chen, A. D. Taylor, J. Homola, B. Hock, and S.Jiang, Sens. Actuators B, 107, 193 (2005).

70）T. Sakai, A. Torimaru, K. Shinahara, N. Miura, T.Imato, K. Toko, and K. Matsumoto, Sens. Mater., 15, 439(2003).

71）D. R. Shankaran, T. Kawaguchi, S. J. Kim, K.Matsumoto, K. Toko, and N. Miura, Anal. Bioanal.Chem., 386, 1313 (2006).

72）M. Kobayashi, M. Sato, Y. Li, N. Soh, K. Nakano, K.Toko, N. Miura, K. Matsumoto, A. Hemmi, Y. Asano,and T. Imato, Talanta, 68, 198 (2005).

73）T. Onodera, K. Miyahara, M. Iwakura, K. Hayashi, N.Miura, K. Matsumoto, and K. Toko, IEEJ Trans. SM, 126(2006) available online.